The intestinal epithelium is constantly renewed by a pool of multipotent intestinal epithelial stem cells (IESCs) located at the crypt base. IESCs are defined by their a) competence to self-renew, and b) multipotency to give rise to all terminally differentiated intestinal epithelial lineages - characteristics otherwise known as 'stemness'. A major question in IESC biology is what controls stemness. Members of the Sox (Sry Box) family of transcription factors function as master control switches for stemness in other tissues. This proposal aims to define the specific roles and gene targets of Sox9 in IESC stemness. We recently demonstrated that distinct levels of Sox9 differentially mark functional IESCs, progenitors, enteroendocrine cells and Paneth cells. Sox9 and Lgr5 both mark IESCs that are intercalated between and make intimate contact with Paneth cells. New evidence suggests that Paneth cells serve as 'nurse'cells and are critical for IESC maintenance. Ablation of Sox9 in intestinal epithelium during embryogenesis results in the loss of Paneth cells and increased proliferation within the crypts of the small intestine and colon. Our new preliminary data on short-term effects of inducible Sox9 ablation in the adult intestinal epithelium confirm crypt hyperproliferation, but also provide novel evidence for additional phenotypic consequences including expansion and displacement of Paneth cels, morphological defects in Paneth cells, mis-expression of goblet biomarker Muc2 in Paneth cells, reductions in crypt-based enteroendocrine cells and goblet cells, and loss of epithelial mono-layer characteristics. Gene microarray on normal small intestine versus intestine from inducible conditional Sox9-mutants indicates that Sox9 controls genes and pathways involved in proliferation, cell adhesion and migration. Since our preliminary studies ablated Sox9 in a pan-epithelial manner, they do not distinguish which phenotypic effects result from the loss of Sox9 in the IESC versus other Sox9-expressing cell lineages, including Paneth cells. The central hypothesis is that Sox9 normally limits IESC proliferation and preserves multipotency by a) intrinsic regulation of specific target genes within IESCs, and b) regulation of Paneth cell specific genes that extrinsically control IESC proliferation or potency by paracrine influences or by restricting Paneth cells to the IESC niche. To test this hypothesis we propose the following aims: 1) to determine the specific roles of Sox9 in IESCs, 2) to determine how Sox9 regulated genes in Paneth cells impacts IESC stemness, 3) to identify specific gene targets and networks regulated by Sox9 in a) IESCs and b) Paneth cells. Successful completion of these studies will lead to novel findings including intrinsic and extrinsic mechanisms controlling IESC behavior. These findings will likely extend to parallel fields of study related to other tissue specific stem cells. PUBLIC HEALTH RELEVANCE: Intestinal epithelial stem cells (IESCs) constantly regenerate the cells lining the small intestine and colon and play an important role in intestinal homeostasis and disease. Understanding the molecular processes that control IESCs is critical to developing the next generation of therapies against intestinal disease. The proposed project will result in the comprehensive study of a very attractive molecular regulator of IESCs, the transcription factor Sox9. We predict that is controlling IESC behavior from within the cell and through neighboring cells. To test this, mice that are deficient for or over- express Sox9 specifically in IESCs will be generated and characterized. These studies will demonstrate the stem cell-specific roles of Sox9 and demonstrate how the gene regulates IESCs intrinsically. Advanced molecular biology techniques will be utilized to identify genes that Sox9 interacts with, allowing the research team to develop a clear picture of the regulatory network that Sox9 participates in. This will lead to a better understanding of genetic control of IESCs. Additionally, mice lacking Sox9 in Paneth cells, which have been proposed to be "nurse cells" for IESCs, will be characterized for their impact on IESC stemness. This aim will identify extrinsic roles Sox9 plays in regulating IESCs, an approach that is conceptually novel to stem cell biology. As a whole, the projects proposed in this grant will result in a more detailed understanding of basic IESC biology through the study of a powerful regulatory gene and advance the knowledge necessary to develop new therapies against disease of the small intestine and colon.